Neutralizing antibodies specifically binding to novel coronavirus or mutant strain spike protein ntd epitope and preparation method and application thereof

Abstract

The application provides a neutralizing antibody specifically binding to an epitope of an NTD of a spike protein of a novel coronavirus or a mutant strain thereof, and a preparation method and application thereof. The heavy chain CDR1 of the neutralizing antibody comprises an amino acid fragment as shown in SEQ ID NO. 1, the heavy chain CDR2 comprises an amino acid fragment as shown in SEQ ID NO. 2, and the heavy chain CDR3 comprises an amino acid fragment as shown in SEQ ID NO. 3; the light chain CDR1 of the neutralizing antibody comprises an amino acid fragment as shown in SEQ ID NO. 4, the light chain CDR2 comprises an amino acid fragment as shown in SEQ ID NO. 5, and the light chain CDR3 comprises an amino acid fragment as shown in SEQ ID NO. 6. The neutralizing antibody can specifically bind to the NTD domain on the spike protein, can be simultaneously applied to the infection prevention and control of the novel coronavirus and the mutant strain thereof, has broad spectrum, and has high neutralizing activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a neutralizing antibody specifically binding to an epitope of NTD of a spike protein of a novel coronavirus or a mutant strain thereof, and a preparation method and application thereof. BACKGROUND

[0002] The novel coronavirus (SARS-CoV-2) is a positive-strand RNA virus with an envelope. The genome of SARS-CoV-2 is 29.9 kb long, and the genome is a single-stranded, positive-strand RNA. The 5' end of the genome has a cap structure, followed by 6-10 open reading frames (ORFs). The first reading frame accounts for 2 / 3 of the genome and encodes a replicase. The last 1 / 3 of the genome mainly encodes structural proteins, which generally include spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). Among them, the S protein is a transmembrane protein and is highly glycosylated, consisting of 1273 amino acids, with 21-35 glycosylation sites at the N terminus. The S protein forms a special crown structure on the surface of the virus in the form of a trimer, and the coronavirus is thus named.

[0003] Neutralizing antibody therapy is an important means for preventing and treating diseases caused by various pathogenic infections, and the development of new SARS-CoV-2 neutralizing antibodies is of great clinical significance. SUMMARY

[0004] Therefore, one of the purposes of the present application includes providing a new neutralizing antibody specifically binding to an epitope of NTD of a spike protein of a novel coronavirus or a mutant strain thereof, to provide a new technical solution for preventing and treating the novel coronavirus.

[0005] The technical solution for achieving the above purpose of the present application includes:

[0006] In a first aspect of the present application, a neutralizing antibody specifically binding to an epitope of NTD of a spike protein of a novel coronavirus or a mutant strain thereof is provided, wherein the heavy chain CDR1 of the neutralizing antibody comprises an amino acid fragment as shown in SEQ ID NO. 1, the heavy chain CDR2 comprises an amino acid fragment as shown in SEQ ID NO. 2, and the heavy chain CDR3 comprises an amino acid fragment as shown in SEQ ID NO. 3.

[0007] The light chain CDR1 of the neutralizing antibody comprises an amino acid fragment as shown in SEQ ID NO. 4, the light chain CDR2 comprises an amino acid fragment as shown in SEQ ID NO. 5, and the light chain CDR3 comprises an amino acid fragment as shown in SEQ ID NO. 6.

[0008] In some embodiments of the present application, the neutralizing antibody satisfies one or more of the following conditions:

[0009] (1) the heavy chain variable region of the neutralizing antibody is as set forth in SEQ ID NO. 7; and,

[0010] (2) the light chain variable region of the neutralizing antibody is as set forth in SEQ ID NO. 8.

[0011] In some embodiments of the present application, the neutralizing antibody satisfies one or more of the following conditions:

[0012] 1) the species origin of the constant region of the neutralizing antibody is human; and,

[0013] 2) the constant region of the neutralizing antibody is an IgGl constant region.

[0014] In some embodiments of the present application, the neutralizing antibody satisfies one or more of the following conditions:

[0015] (I) the light chain constant region of the neutralizing antibody is as set forth in SEQ ID NO. 9; and,

[0016] (II) the heavy chain constant region of the neutralizing antibody is as set forth in SEQ ID NO. 10.

[0017] In a second aspect of the present application, a recombinant protein is provided, which comprises one or more of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3, the heavy chain variable region and the light chain variable region defined in the first aspect.

[0018] In a third aspect of the present application, a detection reagent, a detection kit or a medicament is provided, comprising the neutralizing antibody of the first aspect or the recombinant protein of the second aspect.

[0019] In a fourth aspect of the present application, a nucleic acid is provided, comprising a nucleic acid segment for encoding the neutralizing antibody of the first aspect or the recombinant protein of the second aspect.

[0020] In a fifth aspect of the present application, a recombinant expression vector is provided, comprising the nucleic acid of the fourth aspect.

[0021] In some embodiments of the present application, the recombinant expression vector is an antibody expression vector.

[0022] In some embodiments of the present application, the vector is selected from the group consisting of a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus or a combination thereof.

[0023] In some embodiments of the present application, the recombinant expression vector is AbVec2.0-IGHG1 and / or AbVec1.1-IGKC expression vector.

[0024] In a sixth aspect of the present application, a host cell comprising the nucleic acid of the fourth aspect or the recombinant expression vector of the fifth aspect is provided.

[0025] In some embodiments of the present application, the host cell is 293T cell or Expi293F cell.

[0026] In a seventh aspect of the present application, a method for preparing the neutralizing antibody of the first aspect or the recombinant protein of the second aspect is provided, the method comprising the following steps:

[0027] culturing the host cell of the sixth aspect, and isolating the antibody or the recombinant protein from the obtained culture.

[0028] In an eighth aspect of the present application, the neutralizing antibody of the first aspect or the recombinant protein of the second aspect is used for preparing a drug for preventing and treating novel coronavirus infection.

[0029] Compared with the prior art, the above technical solutions provided by the present application have at least the following beneficial effects:

[0030] The neutralizing antibody provided by the present application comprises CDRs with specific sequences, can specifically bind to the spike protein of the novel coronavirus or mutant strains thereof, and specifically bind to the NTD domain on the spike protein, thereby preventing the infection of the virus to the cell, and ultimately achieving the protection effect. Moreover, the neutralizing antibody can be simultaneously applied to the infection prevention and control of the novel coronavirus and mutant strains thereof, and has broad spectrum, which is not possessed by the existing antibodies binding to the NTD domain. Meanwhile, the neutralizing antibody has strong targeting property and high neutralizing activity. Overall, the present application provides a new candidate scheme for preventing and treating the novel coronavirus infection. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0032] Figure 1 Figure for the binding activity detection result of the monoclonal neutralizing antibody in Example 2 of the present application;

[0033] Figure 2 Figure 2 shows the results of the neutralization assay of the monoclonal neutralizing antibody of Example 2 against SARS-CoV-2 mutant virus live virus. DETAILED DESCRIPTION

[0034] The present application will be further described with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are intended to illustrate the present application and not to limit the scope of the present application. The purpose of these embodiments and examples is to make the disclosure of the present application more thorough and complete. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or changes without departing from the spirit and scope of the present application, and the equivalent forms thus fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description of the present application herein is for the purpose of describing embodiments and examples only and is not intended to be limiting of the present application.

[0036] Terminology

[0037] Unless otherwise indicated or unless the context clearly indicates otherwise, the terms or phrases used herein have the following meanings:

[0038] The selection range of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, which includes any two relevant listed items, any more relevant listed items, or all relevant listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0039] In the present application, "multiple", "various", "multiple times", "multiple" and the like, if not specifically limited, refer to greater than or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0040] As used herein, "combinations thereof", "any combination thereof", "any combination manner thereof" and the like include all suitable combination manners of any two or more of the listed items.

[0041] As used herein, "suitable", "suitable manner", "any suitable manner" and the like are subject to the ability to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0042] As used herein, "preferably", "better", "better", "preferably" only describe the implementation manner or embodiment with better effect, and it should be understood that it does not constitute a limitation on the protection scope of the present application.

[0043] In the present application, "further", "more further", "particularly" and the like are used for description purposes, indicating differences in content, but should not be understood as a limitation on the protection scope of the present application.

[0044] In the present application, "optionally", "optional", "optional" means optional, i.e. selected from any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, if there is no special description, and there is no contradictory relationship or mutual restriction, each "optional" is independent.

[0045] In the present application, the terms "first", "second", "third", "fourth" and the like in the "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the technical features indicated. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration and description, and should be understood as not constituting a closed limitation on the quantity.

[0046] In the present application, among the technical features described in an open manner, both the closed technical solution consisting of the listed features and the open technical solution containing the listed features are included.

[0047] In the present application, with respect to the numerical interval (i.e. numerical range), if not otherwise specified, the optional numerical distribution within the above-mentioned numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range, as well as every numerical value between the two numerical end points. If not otherwise specified, when the numerical interval only points to the integers within the numerical interval, including the two end point integers of the numerical range and every integer between the two end points, in this document, it is equivalent to directly listing every integer, for example, t is an integer selected from 1 to 10, which means that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this document should be understood to include any and all sub-ranges falling within them.

[0048] In the present application, the temperature parameter, if not otherwise specified, allows for constant temperature treatment, and also allows for fluctuations within a certain temperature interval. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0049] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0050] All the documents mentioned in the present application are cited as references in the present application as if each document is cited as a reference individually. The cited documents are cited in the present application in their entirety, in their entirety purpose, unless and to the extent that the cited documents conflict with the description of the present application and / or the purpose of the present application. When the present application refers to the cited documents, the definitions of the related technical features, terms, names, phrases, etc. in the cited documents are cited in the present application. When the present application refers to the cited documents, the examples, preferred modes of the cited related technical features can be cited in the present application as references, but only to the extent that the present application can be implemented. It should be understood that when the cited content conflicts with the description in the present application, the present application is the correct one or the description in the present application is modified adaptively.

[0051] First aspect of the application

[0052] The present application provides a neutralizing antibody specifically binding to an epitope of NTD of spike protein of novel coronavirus or mutant strain thereof, wherein the heavy chain CDR1 of the neutralizing antibody comprises an amino acid fragment as shown in SEQ ID NO. 1, the heavy chain CDR2 comprises an amino acid fragment as shown in SEQ ID NO. 2, and the heavy chain CDR3 comprises an amino acid fragment as shown in SEQ ID NO. 3.

[0053] The light chain CDR1 of the neutralizing antibody comprises an amino acid fragment as shown in SEQ ID NO. 4, the light chain CDR2 comprises an amino acid fragment as shown in SEQ ID NO. 5, and the light chain CDR3 comprises an amino acid fragment as shown in SEQ ID NO. 6.

[0054] Antibodies and variable regions

[0055] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons, having the same structural core features. An antibody consists of two identical light (L) polypeptides, and two identical heavy (H) polypeptides. Each L polypeptide is linked to a H polypeptide by one covalent disulfide bond and the number of disulfide bonds between the H polypeptides varies among the different immunoglobulin isotypes. There are also interchain disulfide bonds of each H and L polypeptide at regular intervals. At one end of each H polypeptide is a variable region (VH), followed by a number of constant regions. At one end of each L polypeptide is a variable region (VL), and at the other end is a constant region; the constant region of the L polypeptide is opposite the first constant region of the H polypeptide, and the variable region of the L polypeptide is opposite the variable region of the H polypeptide. Particular amino acid residues form an interface between the variable regions of the L and H polypeptides.

[0056] In the present application, the antibody can be monospecific, bispecific, trispecific, or more multispecific.

[0057] In the present application, the antibodies of the present application also include conservative variants thereof, which means polypeptides having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids replaced by similar or closely related amino acids compared to the amino acid sequence of the antibodies of the present application.

[0058] The present application includes not only intact antibodies, but also fragments of the antibodies that are immunologically active or fusion proteins of the antibodies with other sequences. Thus, the present application also includes fragments, derivatives, and analogs of the antibodies.

[0059] In the present application, antibodies include murine, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, which include both human and non-human portions, can be obtained by standard DNA recombination techniques, and are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region from a murine monoclonal antibody and a constant region from a human immunoglobulin (see, e.g., U.S. Patent No. 4,816,567 and U.S. Patent No. 4,816,397, both of which are incorporated herein by reference in their entireties). Humanized antibodies are antibodies derived from non-human species that have one or more complementarity determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0060] As used herein, the term "variable" refers to certain portions of the variable region of an antibody that differ in sequence among antibodies and are responsible for binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable regions of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions within the variable region of the light chain and the variable region of the heavy chain. The more conserved portions of the variable region are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, largely β-sheet in structure, connected by three CDRs, which form loops that connect, and in some cases pack against, the FRs of the same chain and contribute to the overall structure of the variable region. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions of the antibodies are not directly involved in the binding of antibodies to antigen, but exhibit diverse effector functions, such as participation in antibody-dependent cellular cytotoxicity.

[0061] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant regions. Depending on the amino acid sequences of their heavy chain constant regions, immunoglobulins can be assigned to different classes, there are mainly five: IgA, IgD, IgE, IgG, and IgM, which can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally (see, for example, Basic and Clinical Immunology (1994) 7th Ed., Daniel E. Atlas, ed., Appleton & Lange, Norwalk, CT).

[0062] Generally, the antigen binding properties of an antibody can be described by three specific regions on the variable regions of the heavy and light chains, called complementarity determining regions (CDRs), which are interspersed with four framework regions (FRs) whose amino acid sequences are relatively conserved and do not directly participate in binding reactions. The CDRs form loops or complementarity determining regions that are brought together in the tertiary structure of the antibody and are positioned by the FR regions to which they are attached. The CDRs on the heavy chain and the CDRs on the light chain make up the antigen binding site of an antibody. The identification of the FR and CDR regions can be determined by comparing the amino acid sequences of antibodies of the same class or subclass.

[0063] Production of antibodies

[0064] Fragments of the DNA molecules of the antibodies or antigen binding fragments thereof of the present application can be obtained using conventional techniques, such as PCR amplification or screening of genomic libraries. In addition, the coding sequences for the light and heavy chains can be fused together to form a single chain antibody. Once the relevant sequence information is available, the sequence fragments can be obtained in large quantities using recombinant methods. This is typically done by cloning the sequence fragments into vectors, which are then introduced into cells, and the sequence fragments are isolated from the propagated host cells using conventional methods.

[0065] In addition, the sequence fragments can be synthesized using artificial synthesis methods, especially when the fragments are relatively short. Typically, longer sequence fragments are obtained by first synthesizing a number of smaller fragments, which are then ligated together.

[0066] At present, it is possible to obtain the DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present application entirely by chemical synthesis. The DNA sequences can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the present application by chemical synthesis.

[0067] The present application also relates to vectors comprising the appropriate DNA sequences described above and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express the proteins.

[0068] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include, but are not limited to, CHO-S, HEK-293 cells.

[0069] Generally, the transformed host cell is cultured under conditions suitable for expression of the antibody of the present application. The antibody of the present application is then purified using conventional immunoglobulin purification methods commonly used by those skilled in the art, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, or affinity chromatography.

[0070] The resulting monoclonal antibodies are identified using conventional methods. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0071] The antibodies of the present application can be expressed intracellularly, or on the cell membrane, or secreted from the cell. If desired, the recombinant proteins can be isolated and purified using various separation methods based on their physical, chemical, and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation procedures, treatment with protein precipitants (salting-out procedures), centrifugation, osmotic shock, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0072] In some examples, the neutralizing antibody satisfies one or more of the following conditions:

[0073] (1) the heavy chain variable region of the neutralizing antibody is as set forth in SEQ ID NO. 7; and,

[0074] (2) the light chain variable region of the neutralizing antibody is as set forth in SEQ ID NO. 8.

[0075] In some examples, the neutralizing antibody satisfies one or more of the following conditions:

[0076] 1) the species origin of the constant region of the neutralizing antibody is human; and,

[0077] 2) the constant region of the neutralizing antibody is an IgGl constant region.

[0078] In some examples, the neutralizing antibody satisfies one or more of the following conditions:

[0079] (I) the light chain constant region of the neutralizing antibody is as set forth in SEQ ID NO. 9; and,

[0080] (II) the heavy chain constant region of the neutralizing antibody is as set forth in SEQ ID NO. 10.

[0081] Second aspect of the application

[0082] The present application provides a recombinant protein comprising one or more of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3, the heavy chain variable region and the light chain variable region defined in the first aspect.

[0083] In some examples, the recombinant protein further comprises a combination of the heavy chain constant region and the light chain constant region defined in the first aspect.

[0084] In some examples, the recombinant protein can further comprise tag fragments that assist the expression and / or purification of the recombinant protein, including but not limited to 6His tag.

[0085] In the present application, the recombinant protein (or polypeptide) includes but is not limited to a fusion protein.

[0086] In the present application, the recombinant protein can be a monomer, a dimer, or a multimer.

[0087] Third aspect of the application

[0088] The present application provides a detection reagent, a detection kit or a medicament comprising the neutralizing antibody of the first aspect or the recombinant protein of the second aspect

[0089] In the present application, "medicament" refers to any compound having a desired biological activity and having a reactive functional group so as to prepare the conjugate of the present application. The desired biological activity includes diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals. Thus, the term "medicament" refers to compounds identified by the official compendia, such as the United States Pharmacopeia, and the official compendia for example, the United States official homoeopathic pharmacopoeia, the official national formulary, or any supplement to the same. Typical medicaments are listed in the Physicians' Desk Reference (PDR) and the Orange Book of the United States Food and Drug Administration (FDA). It should be understood that as new medicaments are discovered and developed, they should also be included in the "medicament" of the conjugate of the present application.

[0090] Fourth aspect of the application

[0091] The present application provides a nucleic acid comprising a nucleic acid fragment for encoding the neutralizing antibody provided in the first aspect or the recombinant protein defined in the second aspect.

[0092] Fifth aspect of the application

[0093] The present application provides a recombinant expression vector comprising the nucleic acid provided in the fourth aspect.

[0094] Optionally, the recombinant expression vector is an antibody expression vector. The present application does not particularly limit the antibody expression vector, including but not limited to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or a combination thereof. Further optionally, the recombinant expression vector is AbVec2.0-IGHG1 and / or AbVec1.1-IGKC expression vector.

[0095] Sixth aspect of the application

[0096] The present application provides a host cell comprising the nucleic acid provided in the fourth aspect or the recombinant expression vector provided in the fifth aspect.

[0097] In some examples, the nucleic acid can be integrated into the genome of the host cell. The present application does not particularly limit the kind of host cell. Including but not limited to CHO cell, COS cell, NSO cell, HeLa cell, BHK cell and HEK293 cell. For example, the host cell is 293T cell or Expi293F cell.

[0098] Seventh aspect of the application

[0099] The present application provides a method for preparing the neutralizing antibody provided in the first aspect or the recombinant protein provided in the second aspect, comprising the following steps:

[0100] Culturing the host cell provided in the sixth aspect, and isolating the antibody or antigen binding fragment thereof or recombinant protein from the obtained culture.

[0101] Eighth aspect of the application

[0102] The present application provides the use of the neutralizing antibody provided in the first aspect or the recombinant protein provided in the second aspect in the preparation of a medicament for preventing and treating Orthopoxvirus infection.

[0103] The definition of medicament refers to the third aspect above.

[0104] In the present application, prevention includes prevention, control, adjuvant therapy, etc. Specific embodiments

[0106] The embodiments of the present application will be described in detail below with examples. It should be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application. The experimental methods in the following examples, if not specified, refer to the guidance given in the present application, and can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0107] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range, if not specifically stated. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0108] Example 1, specificity Monoclonal neutralizing antibodies binding to NTD epitopes of spike protein of SARS-CoV-2 or its mutant strains Construction, expression and purification of expression vectors for monoclonal neutralizing antibodies Specificity

[0109] Monoclonal neutralizing antibodies binding to NTD epitopes of spike protein of SARS-CoV-2 or its mutant strains GFTFSDYY Monoclonal neutralizing antibodies binding to NTD epitopes of spike protein of SARS-CoV-2 or its mutant strains

[0110] Specifically, the heavy chain variable region is shown as SEQ ID NO. 7, the light chain variable region is shown as SEQ ID NO. 8, the light chain constant region is shown as SEQ ID NO. 9, and the heavy chain constant region is shown as SEQ ID NO. 10.

[0111] SEQ ID NO. 1: GFTFSDYY,

[0112] SEQ ID NO. 2: ISSSGSTI,

[0113] SEQ ID NO. 3: ARGGWELRSLAGGYYGMDV,

[0114] SEQ ID NO. 4: QGIRNDL,

[0115] SEQ ID NO. 5: AAS,

[0116] SEQ ID NO. 6: LQHNSYPWT,

[0117] SEQ ID NO. 7:

[0118] EVQLVESGGGLVKPGRSLRLSCAAS ISSSGSTI MSWIRQAPGKGLEWVSY ARGGWELRSLAGGYYGMDV YYADSVRGRFTISRDNAKNSLYLQMNTLRAEDTAVYYC QGIRNDL WGQGTTVTVSS,

[0119] SEQ ID NO. 8:

[0120] DIVMTQTPFTLSASVGDRVTITCRAS AAS GWYQQKPGKAPKCLIY LQHNSYPWT SLLSGV PSRFSGSGSGTEFTLTISSLQPEDFATYYC Specificity FGQGTKLEIK,

[0121] SEQ ID NO. 9:

[0122] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0123] SEQ ID NO. 10:

[0124] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0125] This example Example 2, specificityThe preparation method of the monoclonal neutralizing antibody combined with the NTD epitope of the novel coronavirus or mutant spike protein comprises the following steps:

[0126] I. The nucleotide sequence fragments containing the coding sequences of the heavy chain variable region (SEQ ID NO. 7) and the light chain variable region (SEQ ID NO. 8) of the monoclonal neutralizing antibody are respectively integrated into AbVec2.0-IGHG1 and AbVec1.1-IGKC (the reference literature for the use of the vector is Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann H. J Immunol Methods. 2008 Jan 1; 329(1-2): 112-24. Epub 2007 Oct 31. 10.1016 / j.jim.2007.09.017 PubMed 17996249) containing the constant region sequences of the heavy chain and light chain of human IgG1 antibody, to obtain the recombinant expression vectors capable of expressing the heavy chain and light chain of the target antibody, respectively.

[0127] II. Transfection of cells, expression and purification of the monoclonal neutralizing antibody

[0128] 1. Cell transfection and expression of the monoclonal neutralizing antibody

[0129] The Gibco Expi293F expression system is used, and the transfection operation is carried out according to the instructions. The steps are briefly described as follows:

[0130] Mix 30 μg of the two recombinant expression vectors DNA respectively expressing the heavy chain and light chain of the monoclonal neutralizing antibody with the transfection reagent ExpiFectamine TM , and place it at room temperature for 20 minutes to form a stable complex;

[0131] Then add it to 25.5 mL of Expi 293F cell culture solution with a concentration of 2.9 x 10 6 cells / mL;

[0132] Culture at 37°C, 8% (v / v) CO2, 125 rpm shaker for 20 hours;

[0133] Add the transfection enhancer 1 and the transfection enhancer 2 brought by the Expi293F expression system;

[0134] Continue to be placed in 37℃, 8% (v / v) CO2, 125 rpm shaker for 4 days.

[0135] 2. Purification

[0136] The culture prepared in step 1 was collected and centrifuged at 3000 rpm for 15 minutes to collect the cell culture supernatant, and the antibody was purified using Protein A magnetic beads from Genscript.

[0137] The purification steps are briefly described as follows:

[0138] Mix the Protein A magnetic beads with the cell culture supernatant and combine on a shaker at room temperature for 4 hours;

[0139] Use a magnetic stand to adsorb the magnetic beads, discard the cell supernatant, and wash the magnetic beads 5 times with 1x PBS containing 0.1% (v / v) Tween 20 at pH 7.0;

[0140] Elute with Elution buffer of 0.1M glycine at pH 2.0;

[0141] Equilibrate with 1M Tris buffer at pH 8.5;

[0142] Take the equilibrated monoclonal neutralizing antibody for desalting and DPBS solvent replacement.

[0143] Store the purified neutralizing antibody in a -80℃ refrigerator.

[0144] Functional analysis of neutralizing antibodies Neutralizing antibodies specific to the NTD epitope of the spike protein of the novel coronavirus or its mutant strains Figure 1 Figure 1

[0145] 1. Detection of the binding activity of the human-specific neutralizing antibody prepared in Example 1 to the NTD epitope of the spike protein of the novel coronavirus or its mutant strains

[0146] The ability of the monoclonal neutralizing antibody to bind to the NTD domain on the spike protein of SARS-CoV-2 virus and its mutant strains was determined by ELISA.

[0147] The steps are briefly described as follows:

[0148] (1) Coat 25 ng of NTD protein (Novoprotein, DRA45) on the SARS-CoV-2 spike protein per well on an ELISA plate with DPBS (Dulbecco's Phosphate Buffered Saline) as the coating solution at 4℃ overnight;

[0149] (2) 10% (v / v) calf serum in DPBS as blocking solution, blocking at 37℃ for 2 hours; adding serially diluted monoclonal neutralizing antibody prepared in Example 1, incubating at 37℃ for 2 hours;

[0150] (3) adding 1:40000 diluted HRP-conjugated Goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch) as secondary antibody, incubating at 37℃ for 1 hour;

[0151] (4) coloring with TMB one-component coloring solution, and then stopping the reaction with 2M sulfuric acid, and detecting the absorbance A450 value with an enzyme-labeled instrument.

[0152] Results:

[0153] The results of the binding activity detection of the monoclonal neutralizing antibody and the antigen are shown in Table 1. Figure 2 It can be seen from Table 1 that the binding activity of the monoclonal neutralizing antibody to the NTD domain of the Spike protein is EC50 = 14.55 μg / mL. Figure 2

[0154] 2. Neutralizing activity detection of human-specific neutralizing antibodies specifically binding to NTD epitopes of novel viruses or mutant strains of spike proteins of SARS-CoV-2 and mutant strains of live viruses

[0155] The SARS-CoV-2 live virus neutralization experiment was performed using SARS-CoV-2 live virus wild type (Wild Type, WT) SARS-CoV-2 / human / CHN / IQTC01 / 2020, Alpha mutant strain, Beta mutant strain, Eta mutant strain, Omicron BA.1 mutant strain and Omicron BA.1.1 mutant strain.

[0156] The steps are briefly described as follows:

[0157] After mixing the quantified SARS-CoV-2 virus and the serially diluted monoclonal neutralizing antibody, incubating at 37℃ for 1 hour, then adding to the pre-prepared Vero E6 cell 96-well plate, continuing to culture at 37℃ for 24 hours;

[0158] After the cell plate was fixed with 4% (v / v) paraformaldehyde for 2 hours, the staining operation was performed;

[0159] Permeating with 0.2% (v / v) Triton X-100 at room temperature for 2 minutes;

[0160] ​Cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological Inc) was used as the primary antibody and incubated at 37°C for 1 h to label viral antigens;

[0161] HRP-conjugated Goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch) was used as the secondary antibody and incubated at 37°C for 1 hour;

[0162] KPL TrueBlue Peroxidase substrates (SeraCare Inc) were used as the chromogenic substrate and the color was developed for 5 min;

[0163] The plate was scanned using a CTL ImmunoSpot S6 Ultra reader (Cellular Technology Ltd), and the number of stained viral antigen foci was counted. The virus neutralization activity (expressed as EC50) was also calculated.

[0164] result:

[0165] The results of the live virus neutralization test of monoclonal neutralizing antibodies are shown in ​ ,Depend on ​ It can be seen that this monoclonal neutralizing antibody can broadly neutralize a variety of SARS-CoV-2 virus mutants, and has good neutralizing activity (WT, 3.388μg / mL; Alpha, 3.531μg / mL; Beta, 5.317μg / mL; Eta, 3.827μg / mL; BA.1, 1.401μg / mL; BA.1.1, 2.881μg / mL).

[0166] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0167] The embodiments described above only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. In addition, it should be understood that after reading the above-mentioned teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A neutralizing antibody that specifically binds to the NTD epitope of the spike protein of a novel coronavirus or its mutant strain, characterized in that: The amino acid sequence of the heavy chain CDR1 of the neutralizing antibody is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.3; The amino acid sequence of the light chain CDR1 of the neutralizing antibody is shown in SEQ ID NO.4, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.

6.

2. The neutralizing antibody that specifically binds to the NTD epitope of the spike protein of the novel coronavirus or its mutant strain according to claim 1, characterized in that The neutralizing antibody meets one or both of the following conditions: (1) the amino acid sequence of the heavy chain variable region of the neutralizing antibody is shown in SEQ ID NO. 7; and, (2) The amino acid sequence of the light chain variable region of the neutralizing antibody is shown in SEQ ID NO.

8.

3. A neutralizing antibody that specifically binds to the NTD epitope of the spike protein of a novel coronavirus or its mutant strain according to claim 1 or 2, characterized in that: The neutralizing antibody meets one or both of the following conditions: 1) The species origin of the constant region of the neutralizing antibody is human; and, 2) The constant region of the neutralizing antibody is an IgG1 constant region.

4. The neutralizing antibody that specifically binds to the NTD epitope of the spike protein of the novel coronavirus or its mutant according to claim 1 or 2, characterized in that The neutralizing antibody meets one or both of the following conditions: (I) the amino acid sequence of the light chain constant region of the neutralizing antibody is shown in SEQ ID NO.9; and, (II) The amino acid sequence of the heavy chain constant region of the neutralizing antibody is shown in SEQ ID NO.

10.

5. A detection reagent, a detection kit or a drug, characterized in that: Comprising the neutralizing antibody according to any one of claims 1 to 4.

6. A nucleic acid, characterized in that Encodes the neutralizing antibody according to any one of claims 1 to 4.

7. A recombinant expression vector, characterized in that: Comprising the nucleic acid according to claim 6.

8. The recombinant expression vector according to claim 7, characterized in that The recombinant expression vector is an antibody expression vector.

9. The recombinant expression vector according to claim 8, characterized in that The vector is selected from bacterial plasmid, bacteriophage, yeast plasmid, mammalian cell virus, retrovirus or a combination thereof.

10. The recombinant expression vector according to claim 9, characterized in that The mammalian cell virus is an adenovirus.

11. The recombinant expression vector according to claim 9, characterized in that The recombinant expression vector is AbVec2.0-IGHG1 and / or AbVec1.1-IGKC expression vector.

12. A host cell, characterized in that Comprising the nucleic acid of claim 6 or the recombinant expression vector of any one of claims 7 to 11.

13. The host cell according to claim 12, characterized in that The host cells are 293T cells or Expi293F cells.

14. The method for preparing the neutralizing antibody according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: The host cell according to any one of claims 12 to 13 is cultured, and the antibody is isolated from the obtained culture.

15. Use of the neutralizing antibody according to any one of claims 1 to 4 in the preparation of a medicament for treating novel coronavirus infection.

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